Accepted Article
Received Date : 07-Nov-2014
Revised Date : 20-Nov-2014
Accepted Date : 21-Nov-2014
Article type
: Original Article
PRL1 modulates root stem cell niche activity and meristem size through WOX5 and
PLTs in Arabidopsis
Hongtao Ji1, Shuangfeng Wang1,2, Kexue Li1, Dóra Szakonyi3,4, Csaba Koncz3,5 and Xia Li1§
1
The State Key Laboratory of Plant Cell & Chromosome Engineering, Center for Agricultural Research
Resources, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 286
Huaizhong Road, Shijiazhuang, Hebei 050021, P.R. China
2
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, P.R. China
3
Max-Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany
4
Current address: Instituto Gulbenkian de Ciência, Plant Molecular Biology; Oeiras
5
Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6726
Szeged, Temesvári krt. 62, Hungary
§
Correspondence: [email protected]
TEL: +86-311-85871744
Running title: PRL1 modulates root growth
Key words: Arabidopsis thaliana, PRL1, root stem cell niche, root meristem, WOX5, PLT.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/tpj.12733
This article is protected by copyright. All rights reserved.
Accepted Article
SUMMARY
The stem cell niche in the root meristem maintains pluripotent stem cells to ensure a constant
supply of cells for root growth. Despite extensive progress, the molecular mechanisms
through which root stem cell fates and stem cell niche activity are determined remain largely
unknown. In Arabidopsis thaliana, the Pleiotropic Regulatory Locus 1 (PRL1) encodes a
WD40-repeat protein subunit of the spliceosome-activating Nineteen complex (NTC) that
plays a role in multiple stress, hormone and developmental signaling pathways. Here, we
show that PRL1 is involved in the control of root meristem size and root stem cell niche
activity. PRL1 is strongly expressed in the root meristem and its loss of function mutation
results in disorganization of the quiescent center (QC), premature stem cell differentiation,
aberrant cell division, and reduced root meristem size. Our genetic studies indicate that PRL1
is required for confined expression of the homeodomain transcription factor WOX5 in the QC
and acts upstream of the transcription factor PLETHORA (PLT) in modulating stem cell niche
activity and root meristem size. These findings define a role for PRL1 as an important
determinant of PLT signaling that modulates maintenance of the stem cell niche and root
meristem size.
INTRODUCTION
In higher plants, root growth is maintained by coordinating cell proliferation and
differentiation. Arabidopsis thaliana is a model plant with typical allorhiz roots consisting of
three concentric layers (epidermis, cortex, and endodermis) surrounding the stele, which
contains the vascular tissues (Dolan et al., 1993). Root tissue cells are derived from the stem
cell niche, comprised of an inner group of mitotically inactive quiescent center (QC) cells and
outer mitotically active stem cells (van den Berg et al., 1995; Scheres, 2007; Dinneny and
Benfey, 2008). The stem daughter cells divide several times in the proximal meristem, and
then differentiate in the transition zone (Ubeda-Tomas and Bennett, 2010). Thus, root
meristem size is maintained by the balance between cell division and differentiation in the
root meristematic zone.
This article is protected by copyright. All rights reserved.
Accepted Article
In recent decades, an extensive effort has been mounted to understand the molecular
mechanism by which the function of QC and activity of stem cell niche is controlled in
Arabidopsis roots. Several key regulators of QC identity and stem cell niche activity were
identified (Di Laurenzio et al., 1996; Aida et al., 2004; Sarkar et al., 2007). One of these, the
WUSCHEL-RELATED HOMEOBOX5 (WOX5) factor is specifically expressed in the QC
and functions as a chief regulator of QC maintenance and tissue homeostasis in the root
meristem. Loss of WOX5 function was demonstrated to cause terminal differentiation of
distal root stem cells (Sarkar et al., 2007). Other genes controlling the maintenance of QC
identity and root stem cell niche activity include SHORT ROOT (SHR) and SCARECROW
(SCR) that code for putative GRAS transcription factors. SHR is mainly expressed in the stele
and can move to the QC and other surrounding cells to activate SCR expression together with
WOX5 for coordinate regulation of QC identity and the balance between root stem cell
division and differentiation. Mutations of SHR and SCR result in aberrant stem cell niche
morphology and defective root meristem (Di Laurenzio et al., 1996; Helariutta et al., 2000;
Sabatini et al., 2003) indicating that the SHR/SCR pathway regulates QC identity and stem
cell niche activity.
In parallel with the SHR/SCR pathway, QC maintenance and root meristem homeostasis
are controlled by the PLETHORA (PLT) family of AP2-domain transcription factors. PLT1
and PTL2 are required for maintenance of the activity and determine the position of stem cell
niche (Aida et al., 2004; Galinha et al., 2007). PLT1 and PLT2 mediate positioning of the QC
depending on local auxin maximum and regulate stem cell niche activity responding to the
auxin gradient (Blilou et al., 2005; Grieneisen et al., 2007; Dinneny and Benfey, 2008). Both
PLT1 and PLT2 are induced by auxin and exhibit a graded expression in the root meristem
reflecting the distribution of auxin (Aida et al., 2004; Galinha et al., 2007; Grieneisen et al.,
2007). The PIN auxin efflux carriers play a key role in controlling PLT1/PLT2 expression in
the distal root meristem (Blilou et al., 2005; Ding and Friml, 2010). In turn, PLT1/PLT2
regulate root-specific PIN expression and polar localization of PINs (Blilou et al., 2005;
Galinha et al., 2007; Pinon et al., 2013). Thus, a feedback loop between auxin homeostasis
and PLT1/PLT2 expression controls root meristem maintenance. Recently, it was shown that
This article is protected by copyright. All rights reserved.
Accepted Article
the RAC/ROP GTPase activator RopGEF7, which is expressed in an auxin-dependent
manner, is involved in transmission of the auxin signal to PLT1/PLT2 in the QC (Chen et al.,
2011). Other data indicate that WOX5 acts upstream of the PLT1 to regulate auxin-mediated
QC determination and root stem cell niche homeostasis (Ding and Friml, 2010). Maintenance
of quiescence in the QC and root stem cell activity is a complex process, which also
modulated by abscisic acid (ABA), ethylene, jasmonate, and brassinosteroids, and several
metabolic and stress signaling pathways (Zhang et al., 2010; Chen et al., 2011; Hacham et al.,
2011; Takatsuka and Umeda, 2014). Nonetheless, many important modules that link hormone
signaling to the cell cycle machinery and maintenance of root stem cell niche are unknown.
Here, we report on the identification of a meristem changed root 1 (mcr1) mutation, which
reduces root meristem size and stem cell niche activity. The mcr1 mutation proved to be
allelic with the prl1 mutation, which inactivates the Pleiotropic Regulatory Locus 1 (PRL1)
that codes for a conserved WD40-repeat protein subunit of the nuclear spliceosome-activating
Nineteen Complex (NTC) (Koncz et al., 2012). PRL1 was originally identified as an
important pleiotropic regulator of plant responses to sugars, multiple hormones including
auxin, ABA, cytokinin, and ethylene; cold stress and defense responses to bacterial and
fungal pathogens (Németh et al., 1998; Palma et al., 2007). The prl1 mutation results in
transcriptional derepression of glucose-responsive genes, whereas the PRL1 protein interacts
with the Arabidopsis sucrose non-fermenting 1 (SNF1) homologs AKIN10 and AKIN11
(Bhalerao et al., 1999), which are central regulators of cellular energy homeostasis and
signaling (Baena-Gonzalez et al., 2007). PRL1 was reported to function as substrate receptor
of CUL4-ROC1-DDB1-PRL1 (CULLIN4-REGULATORS OF CULLINS-DAMAGED DNA
BINDING 1) E3 ubiquitin ligase involved in the degradation of AKIN10 (Lee et al., 2008).
All prl1 mutant alleles, including mcr1 cause dramatic defects in root development
(Németh et al., 1998; Palma et al., 2007). By studying the underlying mechanism, here we
show that PRL1 functions upstream of PLT1/PLT2 to modulate stem cell niche activity and
root meristem size in Arabidopsis. PRL1 modulates root stem cell niche activity and root
apical meristem (RAM) size by maintaining graded expression of PLT1/PLT2 and expression
This article is protected by copyright. All rights reserved.
Accepted Article
of the downstream effector WOX5 in the QC. Furthermore, PRL1 is required for maintenance
of columella stem cell (CSC) and provascular stem cell (PSC) activities. Collectively, these
results show that PRL1 is necessary for QC maintenance, stem cell niche activity, root
meristem size, and induction of PLT1/PLT2 and WOX5 in Arabidopsis roots.
RESULTS
Isolation of a mutant defective in root meristem size and cell differentiation
To identify novel determinants involved in the control of root meristem activity, a genetic
screen using 3,000 independent T-DNA mutagenized lines (Zuo et al., 2000) was
performed by monitoring with root length and elongation. One short root mutant showing
an altered apical root meristem was named mcr1. As illustrated in Figure 1a, the mcr1
mutant showed a short root phenotype when grown on Murashige and Skoog (MS) medium.
The primary root length and size of the meristem of mcr1 seedlings were substantially
reduced (Figures 1b-d). The number of cells in the meristem, defined as the number of
cortical cells in a file extending from the initial cell adjacent to the QC to the first elongated
cell (Dello Ioio et al., 2007), was obviously decreased in the mcr1 mutant compared to wild
type (Figures 1c and 1d). The number of evenly sized cortical cells in the elongation zone
was also markedly lower in mcr1 roots (Figure 1e). By contrast, the cortical cells in the
meristematic and elongation zones were larger in mcr1 roots than in wild type (Figures 1f
and S1a). These results suggested that the short root phenotype of the mcr1 mutant
reflected changes in the activity of the RAM.
To test whether the mcr1 mutant has reduced root meristematic activity, we measured
the rate of mature epidermal cell production between 3 and 10 DAG. We found that the cell
production rate in both mutant and WT roots was relatively constant over a period of 10
days. Wild type produced approximately 30 cells per day, and the average length of cells
was about 135 μm (Figures S1b and S1c). In sharp contrast, mcr1 produced only about 8
cells per day with an average cell length of about 100 μm (Figures S1b and S1c) suggesting
altered regulation of cell division in the RAM. Furthermore, the size of cortical cells in
mature zone in mcr1 roots was reduced by 36% compared to wild type (Figure S1d).
This article is protected by copyright. All rights reserved.
Accepted Article
Intriguingly, we found that the primary root growth rate in mcr1 declined rapidly and
essentially ceased at four weeks after stratification (Figure S1e). In parallel, the size of
RAM in the mutant decreased sharply, and the RAM became barely visible upon four
weeks (Figure S1f). Taken together, these results indicated that MCR1 is essential for the
maintenance of RAM size and root meristematic activity.
mcr1 is a new allele of prl1
To identify the mcr1 locus, a genomic fragment flanking the left border of the T-DNA
insertion in the mutant was isolated by thermal asymmetric interlaced-polymerase chain
reaction (TAIL-PCR). Subsequent BLAST search with the plant DNA-T-DNA junction
sequence revealed that the T-DNA was inserted into intron 14 of PRL1 (Figures 2a and
S2a). Further assays showed that primary root growth in the mcr1 plants was similar that in
prl1-1 (Figure 2b). To confirm whether the T-DNA insertion in PRL1 was responsible for
the short root phenotype, an allelism test was performed by crossing homozygous mcr1 and
prl1-1 mutants. The short root phenotype and all other phenotypic traits of the F1 offspring
were indistinguishable from those previously described and observed in prl1 (Németh et
al., 1998), demonstrating that the mcr1 mutation represented a new prl1 allele (Figure 2b).
Furthermore, the root length of mcr1 mutant carrying a genomic PRL1-GFP fusion
(gPRL1-GFP) construct introduced into mcr1 by crossing showed similar root length as
WT, indicating genetic complementation of the mcr1 mutation (Figure S2b). Thereafter,
mcr1 was renamed as prl1-9 (Flores-Perez et al., 2010). Upon backcross of prl1-9 with WT
(Col-0), the F2 yielded 717 WT and 262 prl1-9 progeny with short roots indicating a 3:1
segregation (χ2 = 1.66 < 3.841; χ2 test with one degree of freedom). Reverse transcription
(RT)-PCR assays indicated that 3’-region of the truncated prl1-9 allele was transcribed as
expected and described for the prl1-1 mutant (Figure 2c), in which a T-DNA insertion in
exon 15 was previously demonstrated to prevent the production of immunologically
detectable C-terminally truncated PRL1 protein product (Németh et al., 1998).
This article is protected by copyright. All rights reserved.
Accepted Article
The prl1-9 mutant is defective in G1/S and G2/M cell cycle transitions
To investigate whether cell cycle progression in the RAM was altered in prl1-9, we compared
the expression patterns of several cell cycle-related genes in the root tips of prl1-9 and WT
seedlings by quantitative qRT-PCR. Among those tested, the plant-specific cyclins CycD1;1,
CycD2;1, and CycD3;1 play roles in the G1/S phase transition of the cell cycle (Menges et al.,
2005; de Jager et al., 2009). The result showed that transcription of CycD1;1 and CycD3;1
was markedly decreased in prl1-9 root tips compared to WT (Figure 2d), whereas the level of
CycD2;1 was unchanged (Figure 2d). We next analyzed the expression of genes encoding
Kip-related proteins (KRPs), which are inhibitors of cyclin-dependent kinase (CDK) activity
that negatively regulate the G1/S transition (De Veylder et al., 2001). We found that KRP2
was upregulated in prl1-9 (Figure 2d). Expression of the histone H4 gene, which is usually
used as a marker of S phase cells, was elevated in prl1-9 root tips compared with wild type,
whereas the expression of E2Fa, active at the G1/S transition, was slightly decreased in
prl1-9 (Figure 2d).
Next, we examined whether the prl1-9 mutation would also affect the G2/M phase
transition. First, we analyzed the expression level of mitotic cyclin CycB1;1 in prl1-9 by
crossing the mutant with a transgenic line expressing CycB1;1:GUS (Colon-Carmona et al.,
1999; Donnelly et al., 1999). Histochemical staining showed that the GUS activity was
dramatically higher in prl1-9 root meristem compared to WT (Figure 2e), suggesting that
the cell cycle in prl1-9 RAM was slowed down at the G2 to M phase transition. It is known
that CycB1;1 transcription is activated in G2 phase, and CycB1;1 is degraded by the
anaphase-promoting complex/cyclosome activator (APC/C) complex at metaphase (Zheng
et al., 2011), APC/C complex contains at least 11 different subunits (APC1-APC11),
including the catalytic core subunits APC2 and APC11, and among them, activation and
substrate specificity of APC2 and APC11 are regulated by the Fizzy-related (FZR) proteins.
In Arabidopsis, there are three FZR homolog genes (FZR1, FZR2, FZR3) (Bao et al., 2012).
qRT-PCR analysis showed that the transcription levels of FZRs genes were reduced in
prl1-9 (Figure 2f), while the CycB1;1 mRNA level was increased (Figure 2g), consistent
with the GUS staining results. We also examined the transcript levels of plant-specific cell
This article is protected by copyright. All rights reserved.
Accepted Article
cycle kinase genes CDKA;1 and CDKBs (Figure 2g), whose expression is strictly regulated
during the cell cycle and is increased between S and M phase. The expression of CDKA;1,
which is expressed throughout the cell cycle (Vandepoele et al., 2002; Menges et al., 2005),
was increased in prl1-9. Transcript levels of CDKB1;1 and CDKB2;1, which are expressed
from S to early M phase and from G2 to M phase, respectively (Segers et al., 1996; Umeda
et al., 1999; Menges et al., 2002), were also markedly higher in prl1-9 compared to WT
(Figure 2g). We further examined the mitotic index in the RAM of prl1-9 and found that
there were fewer mitotic figures (metaphase, anaphase, and telophase) in the RAM of
prl1-9 than in WT plants (Figure S3). In conclusion, these results indicated that the prl1-9
mutation reduced the expression levels of several G1/S specific transcripts while increasing
the expression levels of G2/M phase-specific marker genes suggesting a potential defect in
G2/M phase transition.
PRL1 is expressed in the RAM of primary roots and affects the control of RAM size
To examine in more details the role of PRL1 in root development, we analyzed the expression
pattern of a PRL1 promoter-GUS reporter (PRL1pro:GUS) during root development. Activity
of PRL1pro:GUS was detectable in radicles of germinating seeds as early as 12 h after
germination (Figure 3a). Strong GUS activity was further detectable in the root apical regions
of germinating seedlings at 2 to 3 days after stratification, especially in the apical meristems
of primary roots (Figures 3b and 3c). In young seedlings, the GUS activity was prominent in
the RAM (Figures 3d-f). The cell- and tissue-specific expression of PRL1 during primary root
growth indicates its role in establishing and maintaining the RAM during root development.
Next, we investigated how auxin treatment affects PRL1 expression in the root.
PRL1pro:GUS seedlings were treated with 0.1 nM and 5 µM indole-3-acetic acid (IAA) as
described previously (Peng et al., 2013). The expression of PRL1pro:GUS in the RAM was
not significantly affected by the application of 0.1 nM IAA at 5 h after treatment, and was
also only marginally reduced by treatment with 5 µM IAA (Figure 4a). This was further
confirmed by qRT-PCR measurements of PRL1 mRNA levels in roots of 6-day-old seedlings
(Figure 4b). In transgenic prl1-9 mutant plants carrying a complementing genomic
This article is protected by copyright. All rights reserved.
Accepted Article
PRL1-GFP fusion (gPRL1-GFP) construct, the GFP fluorescence localized in nuclei of root
cells (Figure 4c) was similarly to WT and only marginally reduced by 5 µM IAA treatment
(Figure 4d). These results indicated that auxin does not modify remarkably transcriptional
and post-transcriptional regulation of PRL1. Nonetheless, the prl1-9 mutation appeared to
reduce auxin-stimulated increase of the root meristem size. Exogenous application of 0.1 nM
IAA to roots of 6-day-old WT and prl1-9 seedlings for 24 h resulted in a 23.2% increase in
the number of root meristem cells in WT roots, but only a 12.5% increase in prl1-9 (Figures
4e and 4f). When treated with 5 µM IAA, the size of the root meristem in WT was reduced
by 14.8% compared to 6.3% in prl1-9 (Figures 4e and 4g). Consequently, these results
indicated that the prl1-9 mutation compromises auxin-dependent control of the root meristem
size.
The prl1-9 mutation alters auxin distribution and PIN expression levels in the roots
To investigate whether the prl1-9 mutation alters normal auxin distribution in the roots, we
compared the expression pattern of auxin-responsive DR5:GUS reporter (Ulmasov et al.,
1997; Blilou et al., 2005) in mutant and WT seedlings. As shown in Figures 5a and 5b, the
expression pattern of DR5:GUS reporter was considerably reduced in the prl1-9 mutant
compared to WT suggesting that the mutation altered auxin maximum in the root apex. To
determine whether the prl1-9 mutation would influence the localization or expression of the
PIN auxin efflux carriers, we generated prl1-9 plants expressing PINs in fusion with
GFP/YFP reporters under the control of their native promoters by genetic crosses. The
activity of PIN1pro:PIN1-YFP was markedly reduced in the vascular tissue (Blilou et al.,
2005; Dello Ioio et al., 2008), but showed an extended pattern in the transition zone proximal
to the stem cell region in prl1-9 compared to WT (Figures 5c and 5d). The expression levels
of PIN3pro:PIN3-GFP and PIN7pro:PIN7-GFP (Friml et al., 2002; Dello Ioio et al., 2008)
were markedly lower in the columella cells, as well as the vascular issues, in prl1-9 roots
(Figures 5e, 5f, 5k, and 5l). Remarkably, the prl1-9 mutation diminished the expression of
PIN4pro:GUS (Figures 5g and 5h) and PIN4 protein (Figures 5i and 5j) in the root stem cell
niche suggesting a potential correlation with altered auxin regulation of cell proliferation in
prl1-9 roots. We also examined the DR5:GUS expression in cotyledons in prl1-9 and found
This article is protected by copyright. All rights reserved.
Accepted Article
that DR5:GUS expression in prl1-9 was also reduced compared with the WT (Figure S4),
indicating that the reduced auxin maximum in root meristem of prl1-9 is not due to the
reduced activity of PIN1, PIN3, PIN4 and PIN7. In accordance with profound inhibition of
root elongation by the prl1-9 mutation, the expression pattern of PIN2pro:PIN2-GFP was
confined to a reduced region of differentiation and elongation zones compared to wild type,
but its pattern was unaffected by the prl1-9 mutation (Figures 5m and 5n). The observed shift
in auxin distribution and missing PIN4 accumulation in the root stem cell niche, along with
inhibition of auxin-dependent changes in the cell number in the prl1-9 mutant, indicated that
PRL1 is required for proper control of RAM maintenance and functioning.
PRL1 confines WOX5 expression in the QC and QC identity
Regulation of activity of root stem cell niche is a crucial determinant of root meristem size
(Aida et al., 2004; Della Rovere et al., 2013). Therefore, we examined how the prl1-9
mutation affects the activity of stem cell niche. To test this, the expression patterns of several
cell type-specific marker genes in the stem cell niche were analyzed. QC25:GUS, which is
specifically expressed in the QC of WT (Sabatini et al., 1999), showed extended expression
in the lower layer of columella initials (termed also columella stem cells, CSC) and in the
upper layer of proximal (provascular) stem cells (PSCs) in the prl1-9 mutant (Figure 6a). In
addition, disorganized QCs were frequently observed. Their frequency was only 8% in wild
type (n = 80; at 6 DAG), whereas in prl1-9 it reached as high as 67% (n = 80; at 6 DAG).
These results clearly indicated that the QC cells were mitotically active in prll-9.
The expression of WOX5 in QC is critical for maintenance of the stem cell niche (Sarkar et
al., 2007). Therefore, we tested whether WOX5 expression was altered by the prl1-9 mutation.
In fact, we observed five times higher WOX5 transcript levels in primary roots of 6-day-old
prl1-9 seedlings compared to WT (Figure 6b). To confirm this finding, we examined the
expression pattern of a WOX5pro:GFP during early development of mutant and WT roots. In
WT, WOX5 expression was confined to the QC cells and it was maintained at a stable level
throughout the first six days of germination (Figure 6c). In comparison, we observed
considerably higher WOX5pro:GFP expression in the QC of prl1-9 already one day after
This article is protected by copyright. All rights reserved.
Accepted Article
germination, and WOX5pro:GFP levels continued to increase up to 6 DAG. More
importantly, ectopic WOX5pro:GFP expression was clearly detectable in the PSC stem cell
layer proximal to the stem cell niche (Figure 6c). As further confirmation, the same pattern of
WOX5 activity was observed using a WOX5pro:GUS reporter (Figure 6d). Remarkably, an
extension of WOX5pro:GFP expression to the QC-adjacent lateral cells was already
observable in prl1-9 embryonic roots (Figure 6e). WOX5 expression was ultimately decreased
and diminished only about four weeks after germination, when root growth ceased (Figure
S5). Taken together, these data demonstrated that a failure to maintain proper WOX5
homeostasis and QC cell-specific expression resulted in abnormal (i.e., increased) RAM
activity in the prl1-9 mutant indicating that PRL1 is required for proper control of the dose of
WOX5 in QC and thereby for maintenance of normal QC.
PRL1 modulates the differentiation of distal and proximal stem cells
The QC is an organizing center that is required for maintenance of initial root cell division
and differentiation (van den Berg et al., 1997). As QC specification was compromised in
prl1-9, we investigated whether CSC and PSC activities were also affected. In WT, a single
layer of CSCs was present between the QC and differentiated columella cells marked by
starch granules (Figure 7a). Whereas in WT only 5 ± 1.4% of cells (n = 80) corresponding
to the CSC layer showed starch granule accumulation, in the prl1-9 mutant 69 ± 4.1% of
cells in this layer accumulated starch (n = 80; t-test, P < 0.05; Figures 7a and 7b).
Nonetheless, the expression of CSC-specific marker J2341 was strongly suppressed in
prl1-9 (Figures 7c and 7d), and accordingly the number of columella cell layers was
reduced (Figures 7e and 7f). This supported the conclusion that PRL1 is required for the
maintenance of CSC activity.
Next, we used propidium iodide (PI) staining to examine whether premature PSC
differentiation occurred in the proximal region of the QC. As shown in Figures 7g and 7h,
one or two PSCs were strongly stained by PI in prl1-9 versus WT plants already at the
embryo stage. More provascular cells were stained starting from 1 to 6 DAG, and the
PI-stained cells expanded toward to the PSCs until nearly all of the PSCs in the proximal
This article is protected by copyright. All rights reserved.
Accepted Article
meristem were stained (1 ± 0.4% in WT, n = 80; 98 ± 0.6% in prl1-9, n = 80; t-test, P < 0.01)
(Figures 7i-l). This result indicated that the PSCs of mutant roots differentiated prematurely
into vascular tissues. Based on these data, we concluded that PRL1 controls the maintenance
and status of both PSC and CSC.
PRL1 modulates stem cell niche activity and meristem size via a PLT1/PLT2 dependent
pathway
The PLT pathway modulates auxin-dependent maintenance of stem cell niche (Sabatini et al.,
2003; Aida et al., 2004). To ascertain the genetic relationship between PRL1 and PLT in
regulating stem cell niche activity, we generated a prl1-9plt1-4plt2-2 triple mutant by
crossing prl1-9 with plt1-4plt2-2 and subsequently analyzed the size of the RAM in prl1-9,
plt1-4plt2-2, and plt1-4plt2-2prl1-9 plants. The size of the root meristem in prl1-9 was
significantly larger than that in plt1-4plt2-2. Notably, the size of the root meristem in the
plt1-4plt2-2prl1-9 triple mutant was identical to that of the plt1-4plt2-2 double mutant
(Figures 8a and 8b) indicating that PRL1 functions upstream of PLT1/PLT2 in the regulation
of RAM size.
To confirm the relationship between PRL1 and PLT1/PLT2, we analyzed the influence of
the prl1-9 mutation on the expression of PLT1 and PLT2 by examining the activities of
PLT1pro:PLT1-GFP and PLT2pro:PLT2-GFP reporters in prl1-9. The protein expression
levels of both PLT1 and PLT2 were remarkably reduced in prl1-9 compared to WT (Figures
8c and 8d), we also examined the transcription level of PLT1 and PLT2 in the mutant and
found that PLT1 and PLT2 genes were also reduced in prl1-9 compared to WT (Figure S6).
The results indicated that PRL1 is required for maintenance of normal PLT1 and PLT2 levels.
To validate this conclusion, we have introduced a PLT2 overexpression construct
35Spro:PLT2-GR into the prl1-9 mutant. As shown in Figure 8e, the size of the meristem in
WT plants expressing 35Spro:PLT2-GR was significantly increased after induction with
dexamethasone (DEX) (Figures 8e and 8f), consistent with a previous report (Galinha et al.,
2007). When treated with DEX, the meristem size of prl1-9 expressing 35Spro:PLT2-GR was
also increased to a level comparable with that seen in WT (Figures 8e and 8f).
This article is protected by copyright. All rights reserved.
Accepted Article
Complementation of the prl1-9 root meristem phenotype by overexpression of PLT2 suggests
that PRL1 acts upstream of PLT1/PLT2 in the regulation of root meristem size.
As WOX5 and PLTs act in concert with SHORT ROOT (SHR) and SCARECROW (SCR) to
control QC identity, we also generated scr-1 prl1-9 and shr-2 prl1-9 double mutants and
analyzed their root meristem sizes. The meristems in both scr-1prl1-9 and shr-2prl1-9 were
significantly smaller than those of individual scr-1, shr-2, or prl1-9 mutants suggesting an
additive effect of these mutations (Figures S7a and S7b). Further examination of
SHRpro:SHR-GFP and SCRpro:SCR-GFP activities in prl1-9 revealed that the expression
patterns of both reporter genes were unaltered (Figures S7c and S7d). This ultimately showed
that PRL1 functions independently of the SHR/SCR pathway in regulating root meristem size.
DISCUSSION
Recent studies have identified several key determinants that specify the stem cell niche and
prevent the differentiation of stem cells in the root stem cell niche. These determinants have
led to the discovery of the PLT dependent pathway, which functions downstream of auxin
and modulates auxin-mediated root meristem control (Aida et al., 2004). However, the
mechanism that modulates the root stem cell niche maintenance is not yet fully understood.
Here, we found that PRL1 is an upstream regulator of the PLT1/PLT2 dependent pathway
that modulates root meristem size and stem cell niche maintenance.
In a genetic screen for novel root meristem mutations we identified the Arabidopsis mcr1
mutant that exhibited defects in the root meristem displaying short roots. The mcr1 mutant
was found to carry a T-DNA insertion in the PRL1 gene (Figure 2a). PRL1 encodes a
WD40-repeat protein subunit of the NTC complex and has long been recognized as a central
regulator of transcription, splicing and numerous plant developmental, hormonal and stress
signaling pathways (Németh et al., 1998). Although it has been noticed that the PRL1
expression level was highest in roots and that the prl1 mutant develop short roots (Németh et
al., 1998), the role of PRL1 in root growth remained so far uncharacterized. We found that
loss of the PRL1 function in the mcr1/prl1-9 mutant caused a substantial reduction in the size
This article is protected by copyright. All rights reserved.
Accepted Article
of the RAM (Figures 1c and 1d). Intriguingly, the number of mature epidermal cells in prl1-9
was much smaller than in WT (Figure S1c). Thus, we speculated that the short root
phenotype of prl1-9 was largely due to reduced cellular proliferation in the root. This
prediction was shown to be correct based on the observation of disturbed cell cycle
progression affecting both G1/S and G2/M transitions in the root meristem of prl1-9 (Figure
2d-f and S3). Taking into consideration that PRL1 was expressed at the highest level in the
meristematic zone of emerging radicals and primary roots (Figure 3), we concluded that
PRL1 is required for proper control cell proliferation in the root meristem, and for root
meristem maintenance.
The distribution and maximum level of auxin determine the identity of the stem cell niche
and differentiation of stem cells in the root meristem (Ding and Friml, 2010). We found that
PRL1 transcript levels are only marginally reduced by auxin (Figures 4a-d). Nonetheless, the
cell division response of the root meristem to low and high level of auxin markedly differs
from wild type in prl1-9 mutant (Figures 4e-g). This supports the conclusion that PRL1
modulates the auxin responsiveness of root meristematic cells. The prl1-9 mutation reduces
the expression levels of PIN1, PIN3, PIN4, and PIN7 auxin efflux carriers (Figures 5c-n). In
particular, abolishment of PIN4 gene and PIN4 protein expression in the prl1-9 mutant could
prominently affect auxin accumulation in the root stem cell niche (Figures 5g-j). In fact, we
found that inactivation of PRL1 results in derepressed expression of WOX5 expression in the
QC, as well as in CSCs and PSCs (Figure 6c), which is normally repressed in an
IAA17-dependent fashion by auxin signaling (Tian et al., 2014). Consistent with the finding
that WOX5 overexpression in the QC stimulates auxin synthesis (Tian et al., 2014), we found
that expression of the auxin stimulated reporter DR5:GUS reduced in the prl1-9 mutant
compared with WT, indicating a decreased auxin maximum. Furthermore, the prl1-9
mutation extended the expression of WOX5 into the cells surrounding the QC and resulted in
premature differentiation of both distal CSCs and PSCs in the root meristem (Figure 6c and
Figure 7i-l). This indicated that PRL1 is required for the maintenance of both QC identity and
stem cell fate.
This article is protected by copyright. All rights reserved.
Accepted Article
It has been established that auxin modulates root meristem size and stem cell niche
maintenance by regulating the expression of PLTs (Blilou et al., 2005; Grieneisen et al., 2007;
Dinneny and Benfey, 2008). Nonetheless, several details of how RAM activity and stem cell
niche are controlled by PLT dependent signaling remained unknown. In this study, we
collected several pieces of evidence demonstrating that PRL1 acts upstream of PLT1/PLT2 to
modulate RAM activity and maintenance of the root stem cell niche. The first piece of
evidence derived from an epitasis analysis of prl1-9 and plt1plt2 mutations. The root
meristem size in the plt1-4plt2-2prl1-9 triple mutant was identical to that in plt1-4plt2-2,
instead of that in prl1-9 (Figures 8a and 8b). Next, we showed that the prl1-9 mutation
reduced PLT1 and PLT2 expression in the root meristematic zone (Figures 8c and 8d), and
that DEX-induced PLT2 overexpression resulted in a rescue of the root meristem size defect
of prl1-9 (Figures 8e and 8f). In combination with the analysis of WOX5 expression in prl1-9,
we thus demonstrated that PRL1 plays an important role in maintaining the identity of the QC
and stem cell activity. Previous studies have shown that WOX5 is specifically expressed in
the QC and that it functions upstream of PLTs in distal stem cell maintenance (Ding and
Friml, 2010). Auxin represses WOX5 expression in the root meristem, which in turn regulates
the expression of PLTs, whose levels determine the fate of distal stem cells (Aida et al., 2004;
Sarkar et al., 2007). Consequently, our study defines PRL1 as upstream regulator of
WOX5-PLT pathway in the control of QC identity and distal stem cell activity. Our data
show so far that PRL1 represses WOX5 expression and activates PLT1/PLT2 activity, which
is essential for maintenance of the QC and distal stem cells. In addition, PRL1 activity is also
required for the maintenance of PSCs. However, it remains an important further question how
inactivation of PRL1 leads to derepression of WOX5, which requires further identification of
PRL1 targets in auxin signaling.
Experimental procedures
Plant materials and growth conditions
The A. thaliana (L.) seeds used in this study were surface-sterilized with 50% (v/v)
commercial bleach for 5 min, followed by five rinses with sterilized water. The seeds were
then plated on agar plates containing MS nutrient mix (PhytoTechnology Laboratories®,
This article is protected by copyright. All rights reserved.
Accepted Article
Overland Park, KS, USA) supplemented with 1% sucrose and 0.8% agar at pH 5.7. Two days
after stratification at 4 °C in the dark, the seeds were germinated at 22 °C under a 16-h
light/8-h dark photoperiod. The wild-type accession used in this study is Columbia-0 (Col-0).
The prl1-1 mutant was described by Németh et al. (1998). The following types of transgenic
seeds were obtained: SHRpro:SHR-GFP; SCRpro:SCR-GFP; plt1plt2 (Aida et al., 2004);
PLT1pro:PLT1-GFP and PLT2pro:PLT2-GFP (Matsuzaki et al., 2010); scr-1 (Di Laurenzio
et al., 1996); shr-2 (Levesque et al., 2006); WOX5pro: GFP (Haecker et al., 2004);
WOX5pro:GUS (Sarkar et al., 2007); 35Spro:PLT2-GR (Galinha et al., 2007); J2341 and
QC25:GUS (Sabatini et al., 1999); CycB1;1:GUS (Colon-Carmona et al., 1999); DR5:GUS
(Ulmasov et al., 1997); PIN1pro:PIN1:YFP (Benková et al., 2003); PIN2pro:PIN2:GFP,
PIN3pro:PIN3:GFP, and PIN7pro:PIN7:GFP (Blilou et al., 2005); and PIN4pro:GUS (Friml
et al., 2002). The prl1-9 mutation was introduced into transgenic lines and wild type (Col-0)
by crossing, and independent homozygous lines carrying the mutations and expressing the
reporter genes were identified by PCR screening in combination with GUS staining or
following GFP and YFP fluorescence.
Construction of PRL1pro:GUS and gPRL1-GFP reporter genes
To construct PRL1pro:GUS, first a 7.9 kb XbaI-SpeI fragment carrying the PRL1 gene was
cloned from pgcPRL16 (Németh et al., 1998) into pBS to yield pBS-PRL1. Next, an
XbaI-BmgBI fragment of the PRL1 gene carrying the 3.5 kb upstream promoter region linked
to sequences of the -UTR and coding region extending to the start of the third exon was
inserted into XbaI-SmaI sites of the promoter test vector pPCV812 upstream of the GUS
(uidA) coding region to yield the binary vector pPCV812-PRL1PROM harbouring the
PRL1pro:GUS reporter construct.
The gPRL1-GFP reporter construct was assembled in multiple cloning steps. The PRL1
cDNA PCR amplified with the XhoIF and HASpe primers was cloned into the SmaI site of
pBS to yield pBS-PRL1-cDNA-HA. -UTR of PRL1 extending from position -62 to the third
exon was isolated from pBS-PRL1 as an MscI-BmgBI fragment to replace an MscI-BmgBI
cDNA fragment of pBS-PRL1-cDNA-HA in pBS-PRL1-2introns-cDNA-HA. The coding
This article is protected by copyright. All rights reserved.
Accepted Article
region of PRL1 gene extending from the ATG codon to a SmaI site replacing the stop codon
was PCR amplified with the primers PSM1 and PSM2 and introduced into the SmaI site of
pBS resulting in pBS-PRL1-SmaI. Next, the SmaI-BglII -fragment of PRL1 gene from
pBS-PRL1-SMA
was
inserted
into
BglII
and
filled-in
SpeI
sites
of
pBS-PRL1-2introns-cDNA-HA to create pBS-PRL1-2introns-cDNA-Sma. The GFP coding
region was PCR amplified with GFP-F and GFP-R primers and inserted into XbaI-SacII sites
of the latter plasmid to create pBS-PRL1-2introns-cDNA-GFP. The pPCV002 binary vector
was modified by introducing an XmaI/SmaI site on an XbaI-BamHI fragment from pODB8
(Louvet et al., 1997). The PRL1 promoter region extending 3.5 kb upstream of the ATG
codon was PCR amplified with primers SexAI and UTR, and upon digestion used for
replacement of BstBI-XmaI fragment of pBS-PRL1 genomic clone, to yield the construct
pBS-PRL1-PROM-UTR. From the latter plasmid the promoter region extending to an XmaI
site just upstream of the ATG codon was inserted by XbaI-XmaI into pPCV002-ODB to
create pPCV002-PRL1-PROM-UTR. Finally, the coding region of PRL1 fusion with the GFP
gene was isolated from pBS-PRL1-2introns-cDNA-GFP and upon fill-in T4 DNA polymerase
was inserted into the SmaI site of pPCV002-PRL1-PROM-UTR to create the gPRL1-GFP
expression cassette in the binary vector pPCV002-PRL1-GFP.
The binary vectors pPCV812-PRL1PROM and pPCV002-PRL1-GFP carrying the
PRL1pro:GUS and gPRL1-GFP reporter genes were transferred by electroporation into
Agrobacterium GV3101 (pMP90RK) and used for transformation of WT and prl1 mutant
plants as described (Koncz and Schell, 1986). The sequences of the gene-specific primers
used are listed in Table S1.
Microscopic studies, auxin treatment, and histochemical GUS-staining
Root tips of seedlings were photographed with a Leica DM750 microscope (Leica
Microsystems, Wetzlar, Germany). The number (root meristem cell number is expressed as
the number of cells in the cortex file extending from the QC to the transition zone) and length
of cortical and mature epidermal cells were analyzed using Photoshop 8.0 (Adobe Systems
Inc., San Jose, CA, USA). For auxin treatment, 6-day-old seedlings were transferred to MS
This article is protected by copyright. All rights reserved.
Accepted Article
medium with and without the specified concentrations of IAA (PhytoTechnology
Laboratories®). Starch granules in the root tips were stained with an I-KI solution for 0.5 min
then mounted on slides with HCG solution (chloroacetaldehyde:water:glycerol = 8:3:1) and
examined immediately. DEX induction for the 35Spro:PLT2-GR line was performed by
transfering 6-day-old seedlings onto solid MS medium supplemented with 2 μM DEX.
Histochemical GUS-staining was performed according to the method of Ji et al. (2014).
qRT-PCR analysis
Total RNA extraction (from 300 excised root tips) and real-time PCR was performed as
described as Ji et al. (2014). UBQ5 (At3g62250) was used as a reference gene. The sequences
of the gene-specific primers used are listed in Table S1.
Immunolocalization assay
The PIN4 immunolocalization assay was performed using the InsituPro robot (Friml et al.,
2002, Zhou et al., 2010). The following antibodies and dilutions were used: anti-PIN4
(1:50) antibody and Alexa Fluor®546 secondary antibody (Molecular Probes®, A10036).
Fluorescent samples were inspected by the Leica SP8 confocal laser scanning microscope.
Confocal imaging and analysis
GFP fluorescence was detected with a 488 nm argon laser (25 mW, 5~10% power). Samples
were scanned at a speed setting of 8 using the linear mode; For PI staining, root tip samples
were cut and immersed in 10 μM PI for 1 min and then washed three times with
phosphate-buffered saline, a 543 nm HeNe laser was used for image acquisition (Leica TCS
SP8).
ACKNOWLEDGEMENTS
We thank Dr. Ben Scheres (Wageningen University, Netherlands), Thomas Laux
(University of Freiburg, Germany) and Klaus Palme (University of Freiburg, Germany) for
sharing their published materials. This study was supported by the National Program on
Key Basic Research Project (grant no. 2012CB114300) and the National Natural Science
This article is protected by copyright. All rights reserved.
Accepted Article
Foundation of China (grant no. 31230050 to X. L.).
SUPPORTING INFORMATION
Figure S1. Root phenotype of the mcr1 mutant.
Figure S2. The mcr1 mutation represented a new prl1 allele.
Figure S3. Mitotic index in the RAM of WT and prl1-9 seedlings.
Figure S4. DR5:GUS expression in prl1-9 cotyledons.
Figure S5. WOX5 expression is diminished in the prl1-9 mutant.
Figure S6. PLT1 and PLT2 gene expression analysis.
Figure S7. PRL1 acts independently of the SHR/SCR pathways.
Table S1. Primers used in this study.
REFERENCES
Aida, M., Beis, D., Heidstra, R., Willemsen, V., Blilou, I., Galinha, C., Nussaume, L.,
Noh, Y.S., Amasino, R. and Scheres, B. (2004) The PLETHORA genes mediate
patterning of the Arabidopsis root stem cell niche. Cell, 119, 109-120.
Baena-González E., Rolland, F., Thevelein, J.M. and Sheen, J. (2007) A central
integrator of transcription networks in plant stress and energy signalling.
Nature. 448, 938-42.
Bao, Z., Yang, H. and Hua, J. (2012) Perturbation of cell cycle regulation triggers plant
immune response via activation of disease resistance genes. Proc. Natl Acad. Sci.
USA, 110, 2407-2412.
Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G.
and Friml, J. (2003) Local, efflux-dependent auxin gradients as a common module
for plant organ formation. Cell, 115, 591-602.
Bhalerao, R.P., Salchert, K., Bako, L., Okresz, L., Szabados, L., Muranaka, T.,
Machida, Y., Schell, J. and Koncz, C. (1999) Regulatory interaction of PRL1
WD protein with Arabidopsis SNF1-like protein kinases. Proc. Natl Acad. Sci.
This article is protected by copyright. All rights reserved.
Accepted Article
USA, 96, 5322-5327.
Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R.,
Aida, M., Palme, K. and Scheres, B. (2005) The PIN auxin efflux facilitator
network controls growth and patterning in Arabidopsis roots. Nature, 433, 39-44.
Chen, M., Liu, H., Kong, J., Yang, Y., Zhang, N., Li, R., Yue, J., Huang, J., Li, C.,
Cheung, A.Y. and Tao, L.Z. (2011) RopGEF7 regulates PLETHORA-dependent
maintenance of the root stem cell niche in Arabidopsis. Plant Cell, 23, 2880-2894.
Chen, Q., Sun, J., Zhai, Q., Zhou, W., Qi, L., Xu, L., Wang, B., Chen, R., Jiang, H., Qi,
J., Li, X., Palme, K. and Li, C. (2011) The basic helix-loop-helix transcription
factor MYC2 directly represses PLETHORA expression during jasmonate-mediated
modulation of the root stem cell niche in Arabidopsis. Plant Cell, 23, 3335-3352.
Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P. (1999) Technical
advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS
fusion protein. Plant J. 20, 503-508.
de Jager, S.M., Scofield, S., Huntley, R.P., Robinson, A.S., den Boer, B.G. and Murray,
J.A. (2009) Dissecting regulatory pathways of G1/S control in Arabidopsis:
common and distinct targets of CYCD3;1, E2Fa and E2Fc. Plant Mol. Biol. 71,
345-365.
De Veylder, L., Beeckman, T., Beemster, G.T., Krols, L., Terras, F., Landrieu, I., van
der Schueren, E., Maes, S., Naudts, M. and Inze, D. (2001) Functional analysis
of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell, 13, 1653-1668.
Della Rovere, F., Fattorini, L., D'Angeli, S., Veloccia, A., Falasca, G. and Altamura,
M.M. (2013) Auxin and cytokinin control formation of the quiescent centre in the
adventitious root apex of Arabidopsis. Ann. Bot. 112, 1395-1407.
Dello Ioio, R., Linhares, F.S., Scacchi, E., Casamitjana-Martinez, E., Heidstra, R.,
Costantino, P. and Sabatini, S. (2007) Cytokinins determine Arabidopsis
root-meristem size by controlling cell differentiation. Curr. Biol. 17, 678-682.
Dello Ioio, R., Nakamura, K., Moubayidin, L., Perilli, S., Taniguchi, M., Morita, M.T.,
Aoyama, T., Costantino, P. and Sabatini, S. (2008) A genetic framework for the
control of cell division and differentiation in the root meristem. Science, 322,
This article is protected by copyright. All rights reserved.
Accepted Article
1380-1384.
Deng, X.W., Matsui, M., Wei, N., Wagner, D., Chu, A.M., Feldmann, K.A. and Quail,
P.H. (1992) COP1, an Arabidopsis regulatory gene, encodes a protein with both a
zinc-binding motif and a G beta homologous domain. Cell, 71, 791-801.
Di Laurenzio, L., Wysocka-Diller, J., Malamy, J.E., Pysh, L., Helariutta, Y., Freshour,
G., Hahn, M.G., Feldmann, K.A. and Benfey, P.N. (1996) The SCARECROW
gene regulates an asymmetric cell division that is essential for generating the radial
organization of the Arabidopsis root. Cell, 86, 423-433.
Ding, Z. and Friml, J. (2010) Auxin regulates distal stem cell differentiation in
Arabidopsis roots. Proc. Natl Acad. Sci. USA, 107, 12046-12051.
Dinneny, J.R. and Benfey, P.N. (2008) Plant stem cell niches: standing the test of time.
Cell, 132, 553-557.
Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K. and
Scheres, B. (1993) Cellular organisation of the Arabidopsis thaliana root.
Development, 119, 71-84.
Donnelly, P.M., Bonetta, D., Tsukaya, H., Dengler, R.E. and Dengler, N.G. (1999) Cell
cycling and cell enlargement in developing leaves of Arabidopsis. Development,
215, 407-419.
Flores-Perez, U., Perez-Gil, J., Closa, M., Wright, L.P., Botella-Pavia, P., Phillips,
M.A., Ferrer, A., Gershenzon, J. and Rodriguez-Concepcion, M. (2010)
Pleiotropic regulatory locus 1 (PRL1) integrates the regulation of sugar responses
with isoprenoid metabolism in Arabidopsis. Mol. Plant, 3, 101-112.
Friml, J., Benková, E., Blilou, I., Wisniewska, J., Hamann, T., Ljung, K., Woody, S.,
Sandberg, G., Scheres, B. and Jürgens, G. (2002a) AtPIN4 Mediates Sink-Driven
Auxin Gradients and Root Patterning in Arabidopsis. Cell, 108, 661-673.
Friml, J., Wisniewska, J., Benkova, E., Mendgen, K. and Palme, K. (2002b) Lateral
relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature,
415, 806-809.
Galinha, C., Hofhuis, H., Luijten, M., Willemsen, V., Blilou, I., Heidstra, R. and
Scheres, B. (2007) PLETHORA proteins as dose-dependent master regulators of
This article is protected by copyright. All rights reserved.
Accepted Article
Arabidopsis root development. Nature, 449, 1053-1057.
Grieneisen, V.A., Xu, J., Maree, A.F., Hogeweg, P. and Scheres, B. (2007) Auxin
transport is sufficient to generate a maximum and gradient guiding root growth.
Nature, 449, 1008-1013.
Hacham, Y., Holland, N., Butterfield, C., Ubeda-Tomas, S., Bennett, M.J., Chory, J.
and Savaldi-Goldstein, S. (2011) Brassinosteroid perception in the epidermis
controls root meristem size. Development, 138, 839-848.
Haecker, A., Groß-Hardt, R., Geiges, B., Sarkar, A., Breuninger, H., Herrmann, M.
and Laux, T. (2004) Expression dynamics of WOX genes mark cell fate decisions
during early embryonic patterning in Arabidopsis thaliana. Development, 131,
657-668.
Helariutta, Y., Fukaki, H., Wysocka-Diller, J., Nakajima, K., Jung, J., Sena, G.,
Hauser, M.T. and Benfey, P.N. (2000) The SHORT-ROOT gene controls radial
patterning of the Arabidopsis root through radial signaling. Cell, 101, 555-567.
Ji H., Liu L., Li K., Xie Q., Wang Z., Zhao X. and Li X. (2014) PEG-mediated osmotic
stress induces premature differentiation of the root apical meristem and outgrowth
of lateral roots in wheat. J. Exp. Bot. 65, 4863-4872.
Koncz, C., Jong, F., Villacorta, N., Szakonyi, D. and Koncz, Z. (2012) The
spliceosome-activating complex: molecular mechanisms underlying the function
of a pleiotropic regulator. Front. Plant Sci. 3,9.
Koncz, C. and Schell J. (1986) The promoter of TL-DNA gene 5 controls the tissue
specific expression of chimaeric genes carried by a novel type of Agrobacterium
binary vector. Mol. Gen. Genet. 204, 383-396.
Lee, J.H., Terzaghi, W., Gusmaroli, G., Charron, J.B., Yoon, H.J., Chen, H., He, Y.J.,
Xiong, Y. and Deng, X.W. (2008) Characterization of Arabidopsis and rice DWD
proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases.
Plant Cell, 20, 152-167.
Levesque. M.P., Vernoux, T., Busch, W., Cui, H., Wang, J.Y., Blilou, I., Hassa n,
H., Nakajima, K., Matsumoto, N., Lohmann, J.U., Scheres, B. and Benfey,
P.N.
(2006)
Whole
This article is protected by copyright. All rights reserved.
genome analysis of
Accepted Article
the SHORT-ROOT developmental pathway in Arabidopsis. PLoS Biol. 4, e249.
Louvet, O., Doignon, F. and Crouzet, M. (1997) Stable DNA-binding yeast vector
allowing high-bait expression for use in the two-hybrid system. BioTechniques 23,
816-819.
Matsuzaki, Y., Ogawa-Ohnishi, M., Mori, A. and Matsubayashi, Y. (2010) Secreted
peptide signals required for maintenance of root stem cell niche in Arabidopsis.
Science, 329, 1065-1067.
Menges, M., de Jager, S.M., Gruissem, W. and Murray, J.A. (2005) Global analysis of
the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple
and highly specific profiles of expression and provides a coherent model for plant
cell cycle control. Plant J. 41, 546-566.
Menges, M., Hennig, L., Gruissem, W. and Murray, J.A. (2002) Cell cycle-regulated
gene expression in Arabidopsis. J.Biol. Chem. 277, 41987-42002.
Nemeth, K., Salchert, K., Putnoky, P., Bhalerao, R., Koncz-Kalman, Z.,
Stankovic-Stangeland, B., Bako, L., Mathur, J., Okresz, L., Stabel, S.,
Geigenberger, P., Stitt, M., Redei, G.P., Schell, J. and Koncz, C. (1998)
Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD
protein, in Arabidopsis. Genes Dev. 12, 3059-3073.
Ortega-Martinez, O., Pernas, M., Carol, R.J. and Dolan, L. (2007) Ethylene modulates
stem cell division in the Arabidopsis thaliana root. Science, 317, 507-510.
Palma, K., Zhao, Q., Cheng, Y.T., Bi, D., Monaghan, J., Cheng, W., Zhang, Y. and Li,
X. (2007) Regulation of plant innate immunity by three proteins in a complex
conserved across the plant and animal kingdoms. Genes Dev. 21, 1484-1493.
Peng, Y., Ma, W., Chen, L., Yang, L., Li, S., Zhao, H., Zhao, Y., Jin, W., Li, N., Bevan,
M.W., Li, X., Tong, Y. and Li, Y. (2013) Control of root meristem size by
DA1-RELATED PROTEIN2 in Arabidopsis. Plant physiol. 161, 1542-1556.
Pinon, V., Prasad, K., Grigg, S.P., Sanchez-Perez, G.F. and Scheres, B. (2013) Local
auxin biosynthesis regulation by PLETHORA transcription factors controls
phyllotaxis in Arabidopsis. Proc. Natl Acad. Sci. USA, 110, 1107-1112.
Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle, T., Malamy, J., Benfey, P.,
This article is protected by copyright. All rights reserved.
Accepted Article
Leyser, O., Bechtold, N. and Weisbeek, P. (1999) An Auxin-Dependent Distal
Organizer of Pattern and Polarity in the Arabidopsis Root. Cell, 99, 463-472.
Sabatini, S., Heidstra, R., Wildwater, M. and Scheres, B. (2003) SCARECROW is
involved in positioning the stem cell niche in the Arabidopsis root meristem. Genes
Dev. 17, 354-358.
Sarkar, A.K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima, K.,
Scheres, B., Heidstra, R. and Laux, T. (2007) Conserved factors regulate
signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature, 446,
811-814.
Scheres, B. (2007) Stem-cell niches: nursery rhymes across kingdoms. Nature reviews.
Mol. Cell Biol. 8, 345-354.
Segers, G., Gadisseur, I., Bergounioux, C., de Almeida Engler, J., Jacqmard, A., Van
Montagu, M. and Inze, D. (1996) The Arabidopsis cyclin-dependent kinase gene
cdc2bAt is preferentially expressed during S and G2 phases of the cell cycle. Plant
J. 10, 601-612.
Takatsuka, H. and Umeda, M. (2014) Hormonal control of cell division and elongation
along differentiation trajectories in roots. J.Exp. Bot. 65, 2633-2643.
Tian, H., Wabnik, K., Niu, T., Li, H., Yu, Q., Pollmann, S., Vanneste, S., Govaerts, W.,
Rolcík, J., Geisler, M., Friml, J. and Ding, Z. (2014) WOX5-IAA17 feedback
circuit-mediated cellular auxin response is crucial for the patterning of root stem
cell niches in Arabidopsis. Mol Plant. 7, 277-289.
Ubeda-Tomas, S. and Bennett, M.J. (2010) Plant development: size matters, and it's all
down to hormones. Curr. Biol : CB, 20, R511-513.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T.J. (1997) Aux/IAA proteins
repress expression of reporter genes containing natural and highly active synthetic
auxin response elements. Plant Cell, 9, 1963-1971.
Umeda, M., Umeda-Hara, C., Yamaguchi, M., Hashimoto, J. and Uchimiya, H. (1999)
Differential expression of genes for cyclin-dependent protein kinases in rice plants.
Plant physiol. 119, 31-40.
van den Berg, C., Willemsen, V., Hage, W., Weisbeek, P. and Scheres, B. (1995) Cell
This article is protected by copyright. All rights reserved.
Accepted Article
fate in the Arabidopsis root meristem determined by directional signalling. Nature,
378, 62-65.
van den Berg, C., Willemsen, V., Hendriks, G., Weisbeek, P. and Scheres, B. (1997)
Short-range control of cell differentiation in the Arabidopsis root meristem. Nature,
390, 287-289.
Vandepoele, K., Raes, J., De Veylder, L., Rouze, P., Rombauts, S. and Inze, D. (2002)
Genome-wide analysis of core cell cycle genes in Arabidopsis. Plant Cell, 14,
903-916.
Zhang, H., Han, W., De Smet, I., Talboys, P., Loya, R., Hassan, A., Rong, H., Jurgens,
G., Paul Knox, J. and Wang, M.H. (2010) ABA promotes quiescence of the
quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary
root meristem. Plant J. 64, 764-774.
Zheng, B., Chen, X. and McCormick, S. (2011) The anaphase-promoting complex is a
dual integrator that regulates both MicroRNA-mediated transcriptional regulation of
cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte
development. Plant Cell, 23, 1033-1046.
Zhou, W., Wei, L., Xu, J., Zhai, Q., Jiang, H., Chen, R., Chen, Q., Sun, J., Chu, J.,
Zhu, L., Liu, C.M. and Li, C. (2010) Arabidopsis Tyrosylprotein sulfotransferase
acts in the auxin/PLETHORA pathway in regulating postembryonic maintenance of
the root stem cell niche. Plant Cell, 22, 3692-3709.
Zuo, J., Niu, Q.W. and Chua, N.H. (2000) Technical advance: An estrogen
receptor-based transactivator XVE mediates highly inducible gene expression in
transgenic plants. Plant J. 24, 265-273.
Figure legends
Figure 1. mcr1 shows reduced root meristem size and stunted root growth.
(a) Phenotype of WT (Col-0) and mcr1 seedlings at 6 DAG. Bar = 16 mm.
(b) Primary root length of WT and mcr1 seedlings from germination to 7 DAG. The data
shown are means ± SD (n = 30).
This article is protected by copyright. All rights reserved.
Accepted Article
(c) Photograph of the root meristematic zone in WT and mcr1 plants at 6 DAG. Bar = 20 μm.
(d) Root meristem cell number in WT and mcr1 plants from 1 to 7 DAG. The data shown are
means ± SD (n = 30).
(e) Cell numbers in the elongation zone of WT and mcr1 plants at 3 and 6 DAG. The data
shown are means ± SD (n = 30).
(f) Cortical cell size in WT and mcr1 plants in the meristem and elongation zones at 6 DAG.
The data shown are means ± SD (n = 30).
Asterisks in (b), (d), (e) and (f) denote significant differences by Student’s t-test compared
with WT (*, P < 0.05; **, P < 0.01).
Figure 2. MCR1 encodes PRL1 and modulates cell cycle progression.
(a) PRL1/MCR1 gene structure. The start (ATG) and stop (TGA) codons are indicated. Black
boxes indicate exons. Lines between boxes indicate introns.
(b) Phenotype analysis of F1 generation of double mutant (mcr1♂×prl1-1♀ and prl1-1
♂×mcr1♀) at 6 DAG. Bar = 1 cm.
(c) RT-PCR analysis of PRL1 expression. P1, P2, P3, and P4 denote the positions of the
primers in (a).
(d), (f), and (g) Quantitative real-time RT-PCR analysis of cell cycle-related gene expression
in prl1-9 mutant root tip. UBQ5 was used as a reference. The values are given as means ± SD
(*, P < 0.05, t-test).
(e) CyclinB1;1:GUS expression in WT and prl1-9 plants at 6 DAG. Bars = 30 μm.
Figure 3. Analysis of the PRL1 expression pattern in root tip.
PRL1pro:GUS transgenic seeds were grown on MS medium for (a) 12 h (Bar = 120 μm), (b)
2 DAG (Bar = 120 μm), (c) 3 DAG (Bar = 150 μm), (d) 5 DAG (Bar = 300 μm), (e) 7 DAG
(Bar = 500 μm), and (f) 12 DAG (Bar = 500 μm) before GUS staining assays.
Figure 4. Auxin influences PRL1 gene and protein expression.
(a) PRL1pro:GUS transgenic seedlings (6 DAG) were treated with 0.1 nM or 5 μM IAA for 5
h before GUS staining assays. Bars = 50 mm.
This article is protected by copyright. All rights reserved.
Accepted Article
(b) Quantitative real-time analysis of auxin-regulated PRL1 expression in wild type.
Seedlings (6 DAG) were treated with 0.1 nM or 5 μM IAA for 5 h. The values given are
means ± SD (*, P < 0.05, t-test).
(c) gPRL1-GFP transgenic seedlings (6 DAG) were treated with 0.1 nM or 5 μM IAA for 5 h,
respectively, before GFP assays.
(d) Fluorescence quantification of auxin-treated gPRL1-GFP from (c). The intensity values
detected by confocal were compared with untreated wild type (set at 1.0). The values given
are means ± SD (*, P < 0.05, t-test).
(e) Root meristem tissues of 6-day-old WT or prl1-9 seedlings treated with 0.1 nM or 5 μM
IAA for 24 h, respectively. Black vertical lines represent the length of the meristem.
(f) and (g) Average number of cortical cells in root meristems of 6-day-old WT or prl1-9
seedlings from (e). The values given in (f) and (g) are means ± SD. Different letters shows
the significant differences with One Way ANOVA analysis (P < 0.05). Bars = 80 μm.
Figure 5. The mutation of PRL1 affects auxin maximum and PINs expression level.
(a) and (b) Expression patterns of the DR5:GUS reporters in WT (a) and prl1-9 (b) plants at 6
DAG.
(c) and (d) PIN1pro:PIN1:YFP expression in WT (c) and prl1-9 (d) plants at 6 DAG.
(e) and (f) PIN3pro:PIN3:GFP expression in WT (e) and prl1-9 (f) plants at 6 DAG.
(g) and (h) PIN4pro:GUS expression in WT (g) and prl1-9 (h) plants at 6 DAG. Arrowheads
denote QC cells.
(i) and (j) The protein level of PIN4 in WT (i) and prl1-9 (j) plants at 6 DAG using
immunohistological method with the PIN4 antibody.
(k) and (l) PIN7pro:PIN7:GFP expression in WT (k) and prl1-9 (l) plants at 6 DAG.
(m) and (n) PIN2pro:PIN2:GFP expression in WT (m) and prl1-9 (n) plants at 6 DAG. Bars
= 100 μm.
Figure 6. prl1-9 affects stem cell niche activity.
(a) Double staining for the QC25:GUS reporter (blue) and starch granules (brown) in WT and
prl1-9 plants at 6 DAG. Bars = 50 μm.
This article is protected by copyright. All rights reserved.
Accepted Article
(b) Quantitative real-time PCR analysis of WOX5 expression in WT and prl1-9 plants. The
values given are means ± SD. Asterisks denote significant differences by Student’s t-test
compared with WT (*, P < 0.05).
(c) WOX5pro:GFP expression pattern in WT and prl1-9 plants at 1, 3 and 6 DAG. Bars = 50
μm.
(d) WOX5pro:GUS expression pattern in WT and prl1-9 plants at 6 DAG. Bars = 50 μm.
(e) WOX5pro:GFP expression pattern in WT and prl1-9 plants at the mature embryo stage.
Bars = 50 μm.
Figure 7. prl1-9 affects distal and proximal stem cell activity.
(a) and (b) I-KI staining of WT (a) and prl1-9 (b) plants at 6 DAG.
(c) and (d) Expression pattern of J2341 in WT (c) and prl1-9 (d) plants at 6 DAG.
(e) and (f) Expression pattern of WOX5pro:GUS in WT (e) and prl1-9 (f) plants at 6 DAG. *
denotes the columella cells.
(g) and (h) PI staining of the radicle in WT and prl1-9 plants at 12 h imbibition in water. *
denotes the differentiated cells.
(i), (j), (k), and (l) PI staining of WT and prl1-9 plants at indicated times. Bars = 50 μm.
Figure 8. PRL1 acts upstream of PLT1/PLT2 to modulate meristem size.
(a) Root meristem size in prl1-9 and plt1-4 plt2-2 single, double, and triple mutants. Red
arrowheads indicate the cortex transition zone. Bars = 100 μm.
(b) Average number of cortical cells in the root meristem of WT, prl1-9, plt1-4 plt2-2, and
plt1-4plt2-2prl1-9 plants at 4 DAG. Meristem cell numbers for the indicated genotypes at 4
DAG. The data shown are means ± SD (n = 30) (*, P < 0.05, t-test).
(c) PLT1pro:PLT1:GFP and PLT2pro:PLT2:GFP expression in WT and prl1-9 root tips at
6 DAG.
(d) Quantification of PLT1pro:PLT1:GFP and PLT2pro:PLT2:GFP fluorescence as shown in
(c). The intensity values detected by confocal were compared with WT (set at 1.0). The
values given are means ± SD (*, P < 0.05, t-test).
(e) Root meristem of 35Spro:PLT2-GR and 35Spro:PLT2-GR;prl1-9 seedlings were treated
This article is protected by copyright. All rights reserved.
Accepted Article
with 0 and 2 μM DEX for 2 days.
(f) Average number of cortical cells in root meristem of 35Spro:PLT2-GR and
35Spro:PLT2-GR;prl1-9 seedlings in (e). The values are given as means ± SD (*, P < 0.05,
t-test) compared with their respective controls. Bars = 100 μm.
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.